Identifying RNA transcript isoforms requires intricate protocols that suffer from various enzymatic biases. Here we design three-dimensional molecular constructs that enable identification of transcript isoforms at the single-molecule level using solid-state nanopore microscopy. We refold target RNA into RNA identifiers with designed sets of complementary DNA strands. Each reshaped molecule carries a unique sequence of structural (pseudo)colours. Structural colours consist of DNA structures, protein labels, native RNA structures or a combination of all three. The sequence of structural colours of RNA identifiers enables simultaneous identification and relative quantification of multiple RNA targets without prior amplification. Our Amplification-free RNA TargEt Multiplex Isoform Sensing (ARTEMIS) method reveals structural arrangements in native transcripts in agreement with published variants. ARTEMIS discriminates circular and linear transcript isoforms in a one-step, enzyme-free reaction in a complex human transcriptome using single-molecule read-out.
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We thank J. Zhu and M. Fletcher for the critical reading of the manuscript and useful suggestions. We thank the Howarth Lab from the University of Oxford for the monovalent streptavidin. U.F.K. acknowledges funding from a European Research Council Consolidator grant (DesignerPores no. 647144) and European Research Council Proof-Of-Concept grant (PoreDetect no. 899538). F.B. acknowledges funding from George and Lilian Schiff Foundation Studentship, the Winton Programme for the Physics of Sustainability Ph.D. Scholarship and St John’s College Benefactors’ Scholarship.
F.B. and U.F.K. are inventors for the ARTEMIS method (United Kingdom patent application no. 2113935.7, in process) submitted by Cambridge Enterprise on the behalf of the University of Cambridge. U.F.K. is a cofounder of Cambridge Nucleomics.
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Bošković, F., Keyser, U.F. Nanopore microscope identifies RNA isoforms with structural colours. Nat. Chem. 14, 1258–1264 (2022). https://doi.org/10.1038/s41557-022-01037-5